Soil improver and process for preparing same



United States Patent 3,186,826 50H. IMPROVER AND PRUCESS F011 PREPARHIGSAME Margarita del Muro de Rendon, Division Del Norte Ave, No. 415-3,Mexico City, Mexico No Drawing. Filed Oct. 9, 1963, Ser. No. 314,857 7Claims. (Cl. 71-6) This application is a continuation-in-part ofapplication Serial No. 201,748, filed June 12, 1962, now abandoned.

The present invention refers to a biochemical soil improver and to aprocess for manufacturing it and more particularly, it is related to aprocess for preparing a biochemical soil improver, which comprises amixture of different strains of microorganisms of the speciesThiabacillusthiooxz'dans, T. thioparus and T. denitrificans, withsulphur added, and to the process for manufacturing said product. Theinvention is also more particularly directed to a product of the abovenature by means of which an improvement is effected on salt-, alkaliorsalt-alkalicontaining soils in order to improve their condition, in ashorter time and with lesser closes than if sulphur alone is used,controlling the excess of alkali, reducing the pH values of the soil andallowing a better assimilation of the nutrients of the soils into aplant.

It is a well known, fact that soil might be considered as aphysical-chemical-biological complex. It is also well known that thereare certain causes which determine the fact that a soil might beclassified as a salt-containing, alkali-containing andsalt-alkali-containing soil, and this classification is based upon theinterchangeable sodium percentage in a soil and upon its percentage ofsalt.

It is also a well known fact by those skilled in the soil art, that thedissociation of the salts in water, gives raise to ions; and that thepresence of one or more ions in the salt, if it is contained in excess,causes the formation of alkaline bases with the consequent increase inthe pH value, which of course avoids the optimum solubilization of agreat part of the nutrients with the consequent prejudice for the plant,because it cannot be correctly nourished.

Agricultural soils having these characteristics, heretofore havereceived mechanical and agricultural engineering treatments, such asdrainage constructions, and soil lixiviations, in order to carry thesalts down to lower layers and to avoid substantial amounts thereof inthe crop layer.

The desirability of applying a solid improver of the acidifying type,prior to Washing of soil to lixiviate the salt, resides in transformingall excess of sodium, calcium and magnesium ions and the like, intosulfates (by oxidation of sulphur to sulphuric acid by the action ofmicroorganisms of the genus Thiobacillus). This allows the removal ofthe above mentioned ions by washing out the excess thereof, previouslytransforming the alkaline bases into sulfates which are easily solublein water and can be washed out of the crop radicule zone.

The main improvement thus achieved is the removal of sodium in the formof sodium sulfate, thereby avoiding the washing of the salt with water,when the solid improver has not been applied, from causing hydrolysis ofsodium ions with the consequent dispersion of the clay and increase inthe pH values of the soil. The water washing without prior applicationof the improver to a soil classified as salt-alkali-containing soil, isable to transform it into an alkali-containing soil because of the abovereason.

Another very important reason to apply a soil improver to the soilsbefore washing thereof, is to allow hydration of the clays by means ofthe hydrogen ions from sulphuric acid formed by the oxidation of sulphurwith the aid of microorganisms of the above strains.

It is a well known fact, and many workers have proved it, that a sulphursupply to the soil, will cause oxidation, when said soils containpredetermined microorganisms of the genus Thiobacillus which transformsthe sulphur into sulphuric acids as the final product of the reactionscharacteristic thereof and permit thereby the acidification of the soil.

Therefore, one of the main objects of the present invention is toprovide a product which will act as a soil improver with an acidifyingcharacter, to be applied to saline, alkaline and saline-alkaline soils,and which will effect said acidification in a very effective manner andwithout the aid of any other acidifying agent.

Another object of the present invention is to provide a soil improver ofthe above character, wherein in a series of carriers, plurality ofstrains of microorganisms of the genus Thiobacillus are included,together with a certain amount of sulphur, to avoid the individualapplication of sulphur to the soil so submitting its action to thedoubtful presence of various necessary strains of the above mentionedThiobacillus genus.

A more detailed object of the present invention is to provide a productof the above character, wherein microorganisms of the speciesThiobacillus thiooxidans, T. thioparus and T. denitrificans are includedaltogether, in order to oxidize the sulphur added with the product, in amore etlicient and complete manner.

Other objects and advantages of the present invention will be obvious toanyone skilled in the art, and still others will appear in the followingdetailed description of several preferred embodiments thereof, whichwill be given together with the reactions involved in the action of themicroorganisms of the present product, to better illustrate the presentinvention.

The equations which represent the reactions effected by Thiobacillusthiooxidans are as follows:

The oxidative action of the organism is expressed by the correspondingoxygen atoms which are included in each one of the above equations. Asit is well known, the source of the energy for the growth of theseorganisms is mainly the oxidation of elemental sulphur, however,thiosulfate and tetrathionate are also readily oxidated.

The above microorganism etfects an optimal growth at a pH of from 2.0 to3.0 and, according to the works of Waksman and Starkey, it isdistinguished from any other known microorganism in that it is able totolerate and to produce extremely high concentrations of sulphuric acid,which is a mineral and non-organic acid.

On the other hand, the equations which express the ireactions effectedby Thiobacillus thioparus are as folows:

Starkeys equation:

Na S O +Na CO 0-) 4S+6O +4H O+4g Q This organism produces oxidativeaction on thiosulphate as well as on elemental sulphur. The organismgrows in a neutral or slightly alkaline medium. From the aboveequations, it can be concluded that this particular microorganism iscapable of producing oxidation of thiosulphate and tetrathionate, andsupplies sulphuric acid as a result of said oxidation and also anabundant production of elemental sulphur.

In view of the above equations, it can be easily seen that by mixing, ina soil improver, microorganisms of T hiobacillus thiooxidans,Thz'obacillus thioparus and T. denitrificans as well as sulphur, a veryefiicient transformation of sulphur into sulphuric acid will beobtained, because'a very suitable production of sulphuric acid fromthiosulfates and tetrathionates will be obtained by the action of Tlziobacillus thioparus while the sulphur existing in the product plusthe sulphur produced by-Thiobacillus tlzioparus will be transformed intosulphuric acid which will be a very efficient acidifying agent for thesoils treated with a product containing those microorganisms. Allsulphur contained in the product and produced by Thiobacillus thioparus,will be further oxidized by T hiabacillus thiooxida ns as abovementioned, so that the resulting production of sulphuric acid will beconsiderably increased and the acidifying capability of this agent willbe consequently increased.

With the addition of these organisms to the prime materials mix, theelemental sulphur produced by the above mentioned reactions will beoxidized again by the intervention of Thiobacillus thiooxidans, formingin this way a continuity of reactions of oxidative character in soilmedium, allowing the modification of the soil to be done in a shorterperiod of time.

Finally, the equations involved in the action of the microorganism TIziobacillus denitrificans are as follows:

Under aerobic conditions:

Under anaerobic conditions:

This particular microorganism grows perferably in a neutral medium and,of course, it is capable of oxidizing elemental sulphur as Well asfliiosulphate, under aerobic and anaerobic conditions.

According to the above equations which represent the reactions involvedin the biochemical activity of each one of the microorganisms, it willbe clearly seen that a product containing mixtures of the threemicroorganisms, will be very effective to perform oxidation of elementalsulphur and of salts containing sulphur, such as the thiosulphate andthe tetrathionate, so that the product of the present invention whichcontains the three strains of microorganisms is highly eifective asacidifying agent for soils containing salt, alkali, or'saltalkali.

In order to obtain raw cultures of these organisms, they were isolatedfrom samples of volcanic soils.

The cultures were finally isolated in a pure form fol lowing methodsknown in the art for the growth of microorganisms of this type, and thefollowing examples give in an illustrative manner, some preferredmethods to isolate the microorganisms from the volcanic soils, but itmust be understood that these examples must not be construed aslimitative but only a illustrative of the present invention. 7 V

A preferred embodiment of the invention provides a composition for itsapplication to the soils, which comprises per ton of the totalcomposition, 2.16 gallons of a mixture of broths containing T.tlziooxidans, T. thiopm'us and T. denitrificans, 0.72 gallon each; about1,500 pounds of sulphur flour; about 300 pounds of peat; about 20 poundsof activated charcoal and about 180 pounds of calcium sulphate,depending of course on the nature and composition of the soil Where thecomposition is to be applied.

' EXAMPLE 1 Thiobacillus thz'ooxidans was isolated following the methoddescribed below:

Phosphoric rock powder and sulphur powder were added to a sample ofvolcanic soil. Water was also added.

to obtain the best conditions for the growth of the organisms.Incubation was started at 25 C. for a suitable period of time,determining the presence of such organisms by means of the usualpotentiometric methods, effected at certain intervals of time.

The presence of this sulphur oxidizing bacterium is a function of thevariations of the pH rate. After days of incubation, in order to proceedto isolate the organism in a pure culture, Starkeys medium, having acomposition as stated in the following lines, was inoculated with onegram of this sample:

Ammoniumsulphate, (NI-I SO gm 0.2 Magnesium sulphate, MgSOJI-I O gm 0.5Calcium chloride, CaCl gm 0.25 Ferrous sulphate, FeSOJH O gm 0.01Precipitated sulphur gms 10,00 Distilled water ml ,1000

Portions of one gram of precipitated sulphur were placed in ten dry 250ml, flasks, covering the bottom of the flasks. ml. of the mineralsolution of the medium were carefully poured in each flask, in such away that the sulphur floated. Afterwards each flask was sterilized witha stream of steam for half an hour and this operation was repeated forthree consecutive days. The incubation time varies from 10 days to 2Weeks at room temperature.

A very pure culture of the microorganism T hiobwcz'llus thiooxidans wasobtained, quite suitable to be used .in the preparation of the soilimprover of the present invention.

EXAMPLE 2 Thiobacillus thz'oparus was also isolated from raw culturesobtained from a volcanic soil sample, by means of the process whichcomprises first preparing a Beijerincks medium, which composition is asfollows:

Sodium thiosulphate, Na S O -5H O gms.. 5.0 Ammonium chloride, NHQCI gm0.1 Sodium bicarbonate, NaHCO gm 1.0 Disodium phosphate, Na HPO -2H O gm0.2 Magnesium chloride, MgCl -6H O gm 0.1 Tap water ml 1000 Sodiumthiosulphate and carbonate are separately sterilized in a small portionof water, and added to the solution of the other salts. Before use, afew drops of sterilized ferrous sulphate was added to the solution. Thissolution is distributed in flasks of 250 ml. (100ml. in each flask) andthese portions are inoculated with one gram of the soil sample. Eachweek the oxidized thiosulphate is determined by means of an iodinesolution according to the recommended standard method. When there isevidence of disappearance of thiosulphate, the organism is observedthrough microscope, verifying the presence of precipitated sulphurgranules.

To prepare pure liquid cultures of the aboveorganism, colonies thereofare transferred by means of inoculation, to a mixture of the threefollowing media as stated below:

The pH of the above mixture is adjusted to 7.8 and 90 ml. portionsthereof are distributed in each one of ten 250 ml. flasks.

Na S O Tap water ml gms 10.0 50

This solution is sterilized separately in flasks.

gm ml 02 4 Tap water This solution is also sterilized separately inflasks.

The above media, before undergoing inoculation, are mixed in theproportions of 90 ml. of medium (1) per 5 ml. of each one of mediums (2)and (3). The time of incubation for the microorganisms is generally offrom days to two weeks at room temperature.

As T hiobacillzrs denitrificans grow aerobically in the absence ofnitrate, but needs nitrate or ammonia when growing anaerobically, inorder to proceed to the isolation thereof, a mixture of soil which wasfertilized with manure was used to inoculate the following medium(Trautwein) In order to proceed to inoculation, the above medium wasdistributed in several tubes.

As a separate operation, the above medium but without the thiosulphateand with 1.5% agar added is charged in flasks of 50 ml. The resultingmixture is poured in plates and then 0.25 gm. of Na S O -5H O is addedper each 50 ml. of the agar thus obtained, from a previously sterilizedstock solution. 1

The tubes having the above medium, were inoculated with 0.5 gram of thesoil sample, and were incubated under anaerobic conditions for 2 weeksat room temperature. At the end of the above incubation period, theamount of oxidized sodium thiosulphate was calculated by theconventional methods of iodine titration.

Afterwards, the inoculated tubes were used to pour 1 ml. of each one ofthem into each one of the plates containing thiosulphate agar, and theresulting mixtures were incubated for a week at room temperature. Thecolonies of Thz'obacillus denilrificans thus obtained are thin, clear,or weakly opalescent.

Once having determined the presence of Thiobacillus denitrificans, thefollowing culture medium is prepared and inoculated with the abovecolonies of the isolated organisms.

NH Cl gm 0.5 MgSO -7H O gm 0.6 KNO gms 4.0 Tap water m1 1000 100 ml. ofthe above solution are treated with 1 gram of sodium thiosulphate andthe solution is distributed in long tubes with 10 ml. of solution pereach tube.

To each one of the above tubes, 2 drops of the following solution areadded aseptically:

FeSO -7H O 1.0 H SO (0.02N) 1000 To each one of the tubes 0.1 ml. of thefollowing solution were added:

gm ml Finally, 0.2 ml. of the following solution were added to each oneof the above tubes:

NaI-ICO Tap water gms ml gms ml 6 EXAMPLE 3 Each one of the isolatedcolonies of microorganisms obtained according to the preceding examples,are separately transferred to larger amounts of each one of the culturemediums suitable for the particular microorganisms, so that eachmicroorganism will be transferred to a volume of 3 lts. of each culturemedium; in order to obtain three concentrates of the organisms, each oneamounting to 3 liters, which are thereafter mechanically mixed togetherwith 100 kilograms of a suitable carrier material which comprises:

Percent Sulphur powder with a purity over a ground to 325 mesh 75 Peatfrom vegetal origin 15 Activated carbon from vegetal origin 1Agricultural gypsum (calcium sulphate) 9 In the operation of mixing eachone of the concentrates having large amounts of the three types ofmicroorganisms to be used for the manufacture of the soil improver ofthe present invention, strict observance of predetermined moistureconditions must be effected in order to avoid possible death of theseveral microorganisms, and particularly 0 of T hiobacillus thiooxidans,which microorganism is very sensitive to desiccation, inasmuch as it hasbeen proved (Journal of Bacteriology, vol. X, No. 2, March 1925) thatthis organism can die in the absence of a substantial amount ofmoisture.

Therefore, the preferred process according to the present invention toeffect this admixture of concentrates with the carrying material is asfollows:

3 lts. of each of the concentrates of Thiobacillus thiooxidarzs,Thiobacillus thioparus and Thiobacillus denitrificans, are mixed by thecommon mechanical methods with the peat, which moisture must lay between14 and 15% throughout the whole period of time of elaboration, in orderthat the organisms will be maintained alive within the optimalconditions of moisture which are necessary.

No sterile conditions need prevail, inasmuch as the great amount ofmicroorganisms present, substantially removed the danger of anycontamination.

The activated carbon and the agricultural gypsum were added then to themixture and finally the sulphur was added to complete the formulation ofthe above composition.

The activated carbon is added to this mixture with the purpose ofstimulating the oxidative reactions the Thiobacillus carry out.

The thus obtained product is then ready for packing, shipping andapplication to the soil, at doses which will be determined according tothe analytical data representative of the soil where it is to be used,and in accordance with the type of soil, whether it besalt-containing,alkali-containing, or salt-alkali-containing, and alsodepending on the base interchange rate and on the pH of the soil.

The following example is given to illustrate a series of tests elfectedin order to show the action of the soil improver of the presentinvention on the particular type of soil, and its influence on the plantgrowth in that particular soil.

EXAMPLE 4 Five plots of salt-alkali containing soil from theagricultural region of Nuevo Leon, Mexico, were placed in wood boxes andtwo varieties of wheat were planted in these plots of soil, pertainingto the variety known as Chapingo 53 and Lerma Rojo, respectively. Thetests were developed in Escuela Superior de Agricultura Antonio Narro,Saltillo, Coahuila, Mexico.

In these greenhouse tests, the soils were selectively treated with afertilizer and with the soil improver of the present invention and theresults two months after planting were as follows.

'3' Test 1 (a) Applied fertilizer: 180 lb./ acre (200 kgs./ha.)

No application of soil improver Budding: 36% Height: Chapingo 53, 17 %2"approx. (44 cms.) Lerma Rojo: 19% approx. (50 cms.) Applied fertilizer:180 lb./ acre (200 kgs./ha.) Soil improver: 180 lb./acre (200 kgs./ha.)Budding: 66% Height: Chapingo 53, 26" approx. (66 cms.)

Lerma Rojo: 30%" approx. (78 cms.)

Test 2 No application of soil improver No application of fertilizerBudding: Height: Chapingo 53, 11%" approx. (29 cms.) Lerma Rojo: 9approx. (23 cms.) Soil improver applied: 180 lb./acre (200 kgs/ha.)Fertilizer applied: 180 lb./acre (200 kgs/ha.) Height: Chapingo 53, 36approx. (66 cms.) Lerma Rojo: 30%? approx. (78 cms.) Budding: 66% Soilimprover applied 223 lb./acre (250 kgs/ha.) Fertilizer applied: 223lb./acre (250 kgs./ha.) Height: Chapingo 53, 26% approx. (68 cms.) LermaRojo: 30 approx. (77 cms.) Budding: 87%

It must be understood that all the carrying materials to be mixed withthe strains of T hiobacillus thiooxidans, thioparus and denitrificans,according to the invention, can be varied according to the particularcharacteristics of the soil where they are to be applied and it mustalso be understood that the only material which is not liable to bechanged is sulphur, because sulphur is essential to perform theoxidative action of the microorganisms in order to transform it intosulphuric acid or sulphates, to effect the acidification of soils.

For instance, the peat, can be replaced by any other material having theproperty of maintaining a suitable content of moisture, in order to keepalive the strains of the genus Thiobacillus, inasmuch as thesemicroorganisms, as above mentioned, are very liable to die when in a drymedium. However, any other material having this same particularity ofmaintaining moisture included, can be used instead of peat.

Under certain conditions, the agriculture gypsum (calcium sulphate) mustbe removed from the present formulation, while under certain otherconditions the use of gypsum will be very effective to provide aneffective action of the present soil improver.

In other Words, if the soil to be treated is rich in sodium ions, theaddition of, calcium sulphate to the soil improver is very convenient,in order to provide calcium ions to the clays and fiocculate them,regardless of the hydrogen ions from the sulphuric acid formed in theoxidative actions of the species of Thiobacillus with the sulphur.present. The

reaction effected in the case of a soil rich in sodium ions is asfollows:

(Clay) 2Na++soil improver (Clay) +washings On the contrary, if the. soilis saturated with calcium ions, as the present solution also containsexcess of calcium, it will provide the soil with a butfering power,which will avoid solubilizationaud freeaccess of other ions to thecrops, with damages to the vegetal nourishment. In this case, thecarrier materials with which the improver of the present invention ismanufactured, must not contain calcium sulphate or gypsum in theircomposition, so that no more calcium will be introduced into the soil,and the application of the soil improver thereto will have as a mainobjective the hydration of the clays, by replacing a major portion ofthe calcium present by the hydrogen ions from sulphuric acid formed bythe Cit 8 oxidative reaction of the microorganisms on sulphur. Thereaction effected in this latter case is as follows: Ca++(colloidparticle) +H SO v 2H+ (colloid particle) +CaSO Therefore, the presenceor absence of calcium sulphate in the carrier materials used tomanufacture the soil improver of the present invention, will depend ofcourse on the nature of the soil and more particularly on the existenceor absence or excess of calcium ions or sodium ions, as above described.

When agricultural gypsum is not used, it should be compensated withsulphur in an equivalent percentage.

What I claim is:

1. Composition for reducing the alkalinity of alkaline and saline soils,consisting essentially of (a) a mixture of cultures of bacteria of thespecies Thiobacillus thiaoxidan's, Thiobacillus thioparus andThiobacillus denimficans, in admixture with a carrier material including(b) finely divided sulfur in an amount suflicient to sustain thesulfur-oxidative action of said bacteria, (c) peat containing from about40% to about 50% moisture to preserve the viability of the bacteria, (d)activated carbon of vegetable origin in an amount suificient to catalyzethe sulfur oxidative reactions of said bacteria.

2. The composition of claim 1 which includes further an amount ofcalcium sulfate sufficient to provide calcium ions to be interchangedfor sodium ions contained in the soil to be treated.

3. Composition for reducing the alkalinity of alkaline and saline soils,consisting essentially of (a) a mixture of culturesof bacteria of thespecies Thiobacillus thiaoxidans, Thz'obacillus thioparus andThiobacillus denitrificans, in admixture with a carrier materialconsisting essentially of 75% by weight of finely divided sulfur, byweight of peat containing from about 40% to 50% moisture, 1% by weightof activated carbon of vegetable origin and 9% by weight of agriculturalgypsum.

A composition for reducing the alkalinity of alkaline and saline soilsconsisting essentially of a mixture of 0.72 gallon each of nutrientbroth cultures of each of the species of bacteria: Thz'obacilluszhiooxidans, Thiabacillus tlzzoparus and T hiobacillus denitrificans,1500 pounds of 325-mesh sulfur, 300 pounds of peat containing from about40% to 50% moisture, pounds of activated charcoal of vegetable origin,and 20 pounds of calcium sulfate.

5. Method '-for the preparation of a composition for reducing thealkalinity of alkaline and saline soils, comprising the steps of mixingnutrient broth cultures of bac teria of the species T hiabacillusthiooxz'dans, Thiobacillzts thioparus and Thiobacillus denitrificans,with peat containing from about to moisture, to preserve the viabilityof the baceteria, adding to said mixture an amount of activated carbonsufiicient to catalyze the sulfur oxidative reactions of said bacteria,and finally adding to said mixture finely divided sulfur in an amountsufficient to sustain the sulfur oxidative action of said bacteria.

6. The method of claim 5 in which there is further added to the mixturean amount of calcium sulfate sufficient to provide calcium ions to beinterchanged for sodium ions contained in the soil to be treated.

7. Method for reducing the alkalinity of alkaline and saline soil whichcomprises the step of applying to said soil the composition of claim 1.

DONALL H. SYLVESTER, Primary Examiner.

ANTHONY SCIAMANNA, Examiner.

1. COMPOSITION FOR REDUCING THE ALKALINITY OF ALKALINE AND SALINE SOILS, CONSISTING ESSENTIALLY OF (A) A MIXTURE OF CULTURES OF BACTERIA OF THE SPECIES THIOBACILLUS THIOOXIDANS, THIOBACILLUS THIOPARUS AND THIOBACILLUS DENITRIFICANS, IN ADMIXTURE WITH A CARRIER MATERIAL INCLUDING (B) FINELY DIVIDED SULFUR IN AN AMOUNT SUFFICIENT TO SUSTAIN THE SULFUR-OXIDATIVE ACTION OF SAID BACTERIA, (C) PEAT CONTAINING FROM ABOUT 40% TO ABOUT 50% MOISTURE TO PRESERVE THE VIABILITY OF THE BACTERIA, (D) ACTIVATED CARBON OF VEGETABLE ORIGIN IN AN AMOUNT SUFFICIENT TO CATALYZE THE SULFUR OXIDATIVE REACTIONS OF SAID BACTERIA.
 5. METHOD FOR THE PREPARATION OF A COMPOSITION FOR REDUCING THE ALKALINE AND SALINE SOIL, COMPRISING THE STEPS OF MIXING NUTRIENT BROTH CULTURES OF BACTERIAL OF THE SPECIES THIOBACILLUS THIOOXIDANS, THIOBACILLUS THIOPARUS AND THIOBACILLUS DENTRIFICANS, WITH PEAT CONTAINING FROM ABOUT 40% TO 50% MOISTURE, TO PRESERVE THE VIABILITY OF THE BACETERIA, ADDING TO SAID MIXTURE AN AMOUNT OF ACTIVATED CARBON SUFFICIENT TO CATALYZE THE SULFUR OXIDATIVE REACTIONS OF SAID BACTERIA, AND FINALLY ADDING TO SAID MIXTURE FINELY DIVIDED SULFUR IN AN AMOUNT SUFFICIENT TO SUSTAIN THE SULFUR OXIDATIVE ACTION OF SAID BACTERIA. 